Clinical evidence demonstrates dietary therapy as highly effective and even a cure for epilepsy. Specifically, children treated with a high-fat, lo-carbohydrate ketogenic diet (KD) can achieve and maintain seizure freedom even after weaning off the KD and drugs. Its potential likely extends well beyond epilepsy, with emerging research identifying benefits in other acute and chronic conditions. Yet despite continuous use since the 1920s it is still not possible to predict whether KD therapy will be successful, and its mechanism remains unknown. One of the most striking aspects of KD treatment is its proven efficacy even in medically-refractory seizures - indicating that this diet mobilizes anti-seizure mechanisms distinct from available drug treatments. To gain new insight into the mysteries of KD therapy we will leverage an exclusive opportunity to analyze a singular clinical repository. A team in Stockholm, Sweden, led by Maria Dahlin, MD, PhD, obtained cerebrospinal fluid (CSF) from each of 25 children with refractory epilepsy at two time points: 1) before and 2) at three months of maintenance on KD therapy. Clinical data and outcomes were tracked and an initial neurotransmitter analysis was performed. Due to their extremely unique and precious nature (repeated lumbar punctures in each child) these samples have been frozen and awaiting the right opportunity: novel and plausible hypotheses regarding KD efficacy, and a reliable and comprehensive analysis. Now, after several years of preparation, we propose to test our ongoing hypotheses on purinergic mechanisms of KDs and, simultaneously, perform a complete metabolic profiling of CSF obtained from humans and rats fed control vs. KD. CSF will be analyzed for more than 200 molecules by Metabolon (Durham, NC) using their most sophisticated platform. Our team is working with Metabolon regarding initial metabolomic analysis and pathway mapping of the human samples. We will continue with a detailed metabolomic and statistical analysis, collect rat CSF and blood, perform a parallel metabolic and subsequent statistical analysis on the rat samples, and proceed to detailed comparison, interpretation, hypothesis testing, and target validation. We hypothesize that these studies will 1) determine how a KD changes the metabolic signature of various pathways as well as individual metabolites; 2) determine which changes correlate with KD efficacy in children; 3) determine which baseline characteristics predict efficacy in children; 4) enable comparison between diet-mediated changes in human and rodent CSF fluid. Predicting efficacy could help identify and motivate patients who could achieve the greatest benefits. Furthermore this work will yield targets to pursue regarding key metabolic changes mobilized by a KD. Student training is included in all conceptual and proposed experimental aspects; students have been involved in the planning and initial analysis. This work represents a significant opportunity to benefit numerous disorders by revealing key mechanisms, identifying biomarkers, and validating animal models.